Tau protein hyperphosphorylation during hibernation of golden hamsters: insights from a brain IP-MS study

Alzheimer"s disease (AD) originates from two major brain changes, extracellular plaques of amyloid beta and intraneuronal tau protein accumulations called tangles. Whereas presence of amyloid beta plaques is a defining feature of AD and is necessary for AD diagnosis, cognitive decline correlates better with tau protein accumulation rather than with amyloid plaque load. In healthy conditions, the microtubule-binding region (MTBR) of tau protein binds to microtubules and stabilises the intracellular scaffold that allows for neurons" characteristic shape. Neurofibrillary tangles (NFTs) are composed of highly phosphorylated, misfolded tau protein and are the main cause for neuronal death in AD progression. Nowadays, phosphorylated tau peptides detected in CSF and plasma serve as diagnostic tool for classifying patients with potential AD for disease modifying treatments. Although in tauopathies hyperphosphorylation of tau lies at the core if the pathology, it is a phenomenon that also occurs under physiological conditions. Reversible hyperphosphorylation of tau has been shown during hibernation i.e., in European ground squirrels, and brown bears. Moreover, hibernating bears accumulate tau aggregates during the hibernation period.

We have developed a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry (IP-MS) that allows for detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, carrying up to three phosphate groups. The method can be used for detailed analysis of tau, not only in AD cases but also in research on other tauopathies and animal models.

Here we have analysed tau in golden hamsters. Brain tissue (cortex) was collected from hamsters during hibernation and from those who were not hibernating at the time. The tissue was homogenised and both the soluble (tris(hydroxymethyl)aminomethane (tris)-buffered saline, TBS) fraction and the insoluble (sarkosyl-insoluble, SI) fraction were collected.

Subsequently, IP was performed using two anti-tau antibodies, HT7 and 77G7. After IP, samples were trypsinased overnight. Tryptic peptides were then analysed using LC-MS using internal, heavy-labelled standards.

Preliminary data shows that during hibernation hamsters have increased levels of phosphorylated tau; pTau-217, pTau-231, and pTau231+235 were all increased 3-5 times. However, there was no increase for MTBR peptides, indicating that golden hamsters do not form tau aggregates during hibernation. The data set also provides information on the truncation pattern of tau as well as other post-translational modifications.